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Endocrinology Vol. 144, No. 11 4799-4810
Copyright © 2003 by The Endocrine Society

Cloning, Regulation of Messenger Ribonucleic Acid Expression, and Function of a New Isoform of Pituitary Adenylate Cyclase-Activating Polypeptide in the Zebrafish Ovary

Yajun Wang, Anderson O. L. Wong and Wei Ge

Department of Biology (Y.W., W.G.), The Chinese University of Hong Kong, Hong Kong, China; and Department of Zoology (A.O.L.W.), The University of Hong Kong, Hong Kong, China

Address all correspondence and requests for reprints to: Wei Ge, Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. E-mail: weige{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increasing evidence suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a local factor in the ovary of mammals. In nonmammalian vertebrates, although the expression of PACAP has also been demonstrated in the ovary, the information on its functions and regulation is limited. In the present study, we identified a new type of PACAP, zebrafish (zf)PACAP38-2, from the zebrafish ovary. The precursor of GHRH-zfPACAP38-2 consists of 175 amino acids with only 64% homology with another type of zebrafish PACAP, zfPACAP38-1. RT-PCR analysis detected two messengers of zfPACAP38-2 in the zebrafish ovary. The short product was more abundant, and it encodes zfPACAP38-2 only, whereas the long form codes for both zfPACAP38-2 and GHRH. Using a primary culture of zebrafish follicle cells, we demonstrated that gonadotropin (human chorionic gonadotropin and goldfish pituitary extract) significantly stimulated zfPACAP38-2 expression within 2 h; however, the effect decreased to the control level after 8 h of treatment. The stimulation of zfPACAP38-2 expression by gonadotropin could be mimicked by cAMP analogs and forskolin but suppressed by H89 (10 µM), suggesting the involvement of the cAMP-protein kinase A signaling pathway. We also examined the expression of PACAP receptor VPAC2-R in the zebrafish ovary. Unlike zfPACAP38-2, which showed a trend of increase during follicle development, the expression of VPAC2-R mRNA in the follicles showed no significant stage-dependent variation, and its expression in the follicle cells did not respond to gonadotropin treatment. Our studies further demonstrated that synthetic zfPACAP38-2 stimulated oocyte maturation and increased the expression of follistatin in zebrafish ovarian follicle cells. These results suggest that zfPACAP38-2 is a potential ovarian factor that mediates gonadotropin actions in paracrine/autocrine manners, and its functional roles are likely, to some extent, related to the ovarian activin/follistatin system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY ADENYLATE CYCLASE-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamic tissues for its potent activity to stimulate cAMP production in cultured rat anterior pituitary cells (1). It has been classified as a new member of the vasoactive intestinal peptide (VIP)/secretin/GHRH family of peptides based on its high amino acid sequence homology with VIP. Two forms of PACAP, PACAP38 and PACAP27, have been identified in natural sources, and they are derived from the same precursor and amidated at the C terminus (1, 2). In mammals, the PACAP38 is the dominant form in most tissues. PACAP acts by binding to three G protein-coupled receptors with typical seven-transmembrane domains, namely PAC1-R, VPAC1-R, and VPAC2-R, respectively (3). PAC1-R is specific for PACAP with much lower affinity for VIP (4, 5, 6, 7), whereas VPAC1-R and VPAC2-R have similar binding affinity for PACAP and VIP (8, 9, 10, 11). All three PACAP receptors signal by stimulating adenylate cyclase activity, leading to an increase in the intracellular cAMP levels. In addition, PAC1-R also activates phospholipase C signaling pathway to increase the cytosolic inositol 1,4,5-triphosphate and free Ca2+ levels (12, 13, 14).

In mammals, PACAP has been well documented to have a wide range of functions in both the central nervous system and peripheral tissues including the intestine, lung, adrenal gland, testis, and ovary (15, 16, 17). There has been growing evidence that PACAP plays important roles in the mammalian hypothalamo-pituitary-gonadal axis to regulate reproductive functions. In the anterior pituitary, PACAP has been demonstrated to be involved in the release of gonadotropins and differential expression of gonadotropin subunits (17, 18). In the ovary, gonadotropins strongly induce a transient expression of PACAP and PAC1-R in the rat granulosa cells in vivo and in vitro (19, 20, 21, 22, 23, 24), and PACAP stimulates cAMP accumulation and steroidogenesis in a dose-dependent manner in the cultured granulosa cells (25, 26). The physiological relevance of PACAP in the ovarian steroidogenesis has been further confirmed by immunoneutralization of the endogenous PACAP, which reduces progesterone production by the granulosa-luteal cells (27). PACAP has also been shown to enhance maturation rate of the follicle-enclosed oocytes (28) but delay that of the denuded oocytes (28). These studies, together with the expression of PAC1-R, VPAC1-R, and VPAC2-R in the ovary (22, 23, 29, 30, 31), suggest that PACAP is an important paracrine/autocrine regulator in the mammalian ovary.

As an extremely conserved regulatory peptide, PACAP has also been well studied in fish, the largest group of vertebrates (32). Interestingly, the organization of the PACAP gene in fish (33, 34, 35, 36) and other nonmammalian species (37, 38) is distinct from that of mammals. In fish, PACAP and GHRH are encoded by one single gene, whereas they are encoded by two separate genes in mammals (15). In the zebrafish, a GHRH-PACAP gene has recently been isolated from a genomic library, and its structure has been analyzed. Southern analysis of the genomic DNA revealed a single band, suggesting a single copy of the gene in the zebrafish genome (36). Interestingly, using RT-PCR and rapid amplification of cDNA ends (RACE), we have recently cloned a cDNA for a new type of GHRH-PACAP from the zebrafish ovary, which shows significant sequence variation from the one reported. To avoid confusion, we designate the zebrafish PACAP identified in the present study as zebrafish (zf)PACAP38-2 to distinguish it from the one previously reported (zfPACAP38-1) (36). The present study was undertaken to investigate the following: 1) the expression of the newly identified zfPACAP38-2 and VPAC2-R receptor in the zebrafish ovary, 2) the regulation of zfPACAP38-2 expression by gonadotropin and the signaling mechanism involved, 3) the effect of PACAP on final oocyte maturation, and 4) the potential relationship of PACAP with other intraovarian regulatory systems such as the activin/follistatin system. Because zebrafish is an excellent model for vertebrate development, the present study will provide valuable information about PACAP and its roles in the ovary of vertebrates.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and hormones
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and restriction enzymes were from Promega (Madison, WI) unless otherwise stated. Human chorionic gonadotropin (hCG), 17{alpha},20ß-dihydroxy-4-pregnen-3-one (DHP), and forskolin were purchased from Sigma-Aldrich, and 8-bromo-cAMP (8-Br-cAMP), dibutyryl-cAMP (db-cAMP), and H89 were from Calbiochem (La Jolla, CA). hCG, 8-Br-cAMP, and db-cAMP were first dissolved in water, whereas DHP was first dissolved in ethanol, and forskolin and H89 were first dissolved in dimethylsulfoxide. They were diluted to the desired concentrations with the medium before use. zfPACAP38-2 was synthesized at the HSC Biotechnology Service Centre (University of Toronto, Toronto, Ontario, Canada) using a Novasyn Crystal automated peptide synthesizer (Novabiochem Ltd., Nottingham, UK). Peptide synthesis was conducted on PAL-PEG-PS resin using the continuous-flow Fmoc-peptide chemistry. The purity of the peptide was approximately 93% as revealed by reverse-phase HPLC, and the molecular weight of zfPACAP38-2 was confirmed by atmospheric pressure ionization mass spectrometry. The pituitary extract was prepared from sexually mature preovulatory female goldfish (Carassius auratus). Briefly, five goldfish pituitary glands were homogenized in phosphate buffer followed by centrifugation at 15,000 rpm for 30 min at 4 C. The supernatant was filtered, and the protein content was determined with Bio-Rad (Hercules, CA) protein assay kit. The protein yield was about 300 µg/pituitary.

Animals
Zebrafish (Danio rerio) were purchased from local pet stores and maintained in flow-through aquaria (36 liters) at 26 C on a 14-h light, 10-h dark photoperiod. The fish were fed twice per day with commercial tropical fish food with a supplement of live brine shrimp once or twice per week. All experiments were performed under license from the Government of the Hong Kong Special Administrative Region and endorsed by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong.

Total RNA isolation
Total RNA was isolated from the tissues, ovarian follicles, and cultured follicle cells with Tri reagent (Molecular Research Center, Cincinnati, OH) according to the protocol of the manufacturer and our previous report (39).

Cloning of PACAP and VPAC2-R fragments from the zebrafish ovary
Two pairs of gene-specific primers were designed based on two expressed sequence tag sequences (fc44a01.y1 and fj30e08.x1) from the Washington University (St. Louis, MO) Zebrafish Expressed Sequence Tag Project for cloning cDNA fragments of PACAP, whereas the primers amplifying VPAC2-R were based on the sequence fj56c05.y1 (Table 1Go). One microgram of total RNA from the ovary was reverse transcribed into single-stranded cDNA with SuperScript II (Invitrogen Co., Carlsbad, CA) followed by PCR amplification with the gene-specific primers. Thirty-three cycles were performed using a cycle profile of 30 sec at 94 C, 30 sec at 58 C, and 60 sec at 72 C, followed by a final 10-min extension at 72 C. The PCR products of expected sizes were isolated from the gel and cloned into pBluescript II KS (+) (Stratagene, La Jolla, CA). The cloned PCR fragments were analyzed by sequencing. The same gene-specific primers were also used later to develop semiquantitative RT-PCR assays for assessing the expression of PACAP and VPAC2-R in the ovary and other tissues.


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  		TABLE 1. Primers used in the RT-PCR assays

 
5'- and 3'-RACE of PACAP
Two gene-specific primers for PACAP were designed based on the cloned cDNA fragment and used for 5'- and 3'-RACE to amplify the 5' and 3' cDNA ends using the SMART-RACE cDNA amplification kit (Clontech Laboratories, Inc., Palo Alto, CA). The amplified cDNA ends were cloned into pBluescript II KS (+) through T/A cloning. Sequencing of the cDNA ends was performed on a series of overlapping subclones generated by exonuclease III and mung bean nuclease deletion. Both strands of the cDNA were sequenced with the BigDye Terminator Cycle Sequencing, version 2.0, Ready Reaction kit and analyzed on the Applied Biosystems (Foster City, CA) PRISM 310 genetic analyzer.

Isolation and incubation of zebrafish ovarian follicles
Ovaries were removed from 15–20 female zebrafish after decapitation and placed in a 35-mm culture dish containing 60% Leibovitz L-15 medium (Invitrogen Co.). The full-grown immature follicles (~0.65 mm in diameter) were carefully separated, and the healthy ones were selected and incubated in 24-well plates for the experiments. The follicles were examined microscopically for germinal vesicle breakdown (GVBD), a visible morphological marker for oocyte maturation.

Primary follicle cell culture
The primary culture of zebrafish ovarian follicle cells was performed according to our previous report (39). Briefly, the midvitellogenic follicles from about 20 female zebrafish were isolated, washed with medium 199 (Invitrogen Co.), and incubated in medium 199 supplemented with 10% fetal calf serum (HyClone, Logan, UT) at 28 C in 5% CO2 for 6 d for the follicle cells to proliferate. The proliferated follicle cells were harvested by trypsinization and plated in 24-well plates for 24 h before treatments.

Northern blot analysis
Northern blot hybridization was performed according to our previous study (40). Briefly, total RNA (15–20 µg) from the whole ovary, full-grown immature follicles, and cultured follicle cells was fractionated by electrophoresis in a 1.1% denaturing agarose gel containing 2.2 M formaldehyde, transferred to positively charged nylon membrane (Roche Applied Science, Mannheim, Germany), and UV cross-linked using the GS Gene Linker (Bio-Rad). The blots were hybridized with digoxigenin-labeled antisense RNA probe prepared from the cloned zebrafish VPAC2-R cDNA fragment (274 bp) by in vitro transcription and detected with the chemiluminescent detection kit according to the manufacturer’s instructions (Roche Applied Science).

Validation of semiquantitative RT-PCR assays for zfPACAP38-2, VPAC2-R, follistatin, and ß-actin
Reverse transcription (RT) was performed at 42 C for 2 h in a total volume of 10 µl consisting of 3 µg total RNA, 1x single-strand buffer (Invitrogen Co.), 10 mM dithiothreitol, 0.5 mM each deoxynucleotide triphosphate, 0.5 µg oligo-deoxythymidine, and 100 U SuperScript II (Invitrogen Co.). To optimize the cycle numbers used for semiquantitative PCR analysis, the RT reaction (0.6 µl) from cultured follicle cells was used as template for PCR amplification. The primers used for zfPACAP38-2, VPAC2-R, follistatin, and ß-actin are listed in Table 1Go. To ensure specific amplification of zfPACAP38-2, the primers were designed over the regions that show significant variation from that of zfPACAP38-1. PCR was carried out in a volume of 30 µl consisting of 1x PCR buffer, 0.2 mM each deoxynucleotide triphosphate, 2.5 mM MgCl2, 0.2 µM each primer, and 0.6 U of Taq polymerase on the Thermal Cycler 9600 (Eppendorf, Hamburg, Germany) for various cycles with the profile of 30 sec at 94 C, 30 sec at 56 C for VPAC2-R, follistatin, and ß-actin or 58 C for zfPACAP38-2, and 60 sec at 72 C. The PCR products from different cycles of amplification were visualized on the Gel-Doc 1000 system (Bio-Rad) after electrophoresis on 1.8% agarose gel containing ethidium bromide, and the signal intensity was quantitated with the Molecular Analyst software (Bio-Rad). The cycle numbers that generate half-maximal amplification were used for semiquantitative analysis of gene expression. The specificity of PCR amplification was confirmed by cloning and sequencing.

Data analysis
The mRNA level of each gene was first calculated as the ratio to that of ß-actin, which was amplified as the internal control, and then expressed as the percentage of the control group. The data were analyzed by one-way ANOVA followed by Dunnett’s test using GraphPad Prism 3.0cx for Macintosh (GraphPad Software, San Diego, CA). The data of percentage GVBD were analyzed after arcsin transformation. We performed all of the experiments at least twice to confirm the results using different batches of animals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a new type of PACAP (zfPACAP38-2) expressed in the zebrafish ovary
Two bands of different sizes were obtained in both 5'- and 3'-RACE using gene-specific primers. Sequence analysis reveals that the band of large size contains complete coding regions for both zfPACAP38-2 and GHRH. However, the small product has the complete coding region for zfPACAP38-2 and the region for 13 amino acids of GHRH at the C terminus, but the region (105 bp) coding for the majority of GHRH molecule is deleted (Figs. 1Go and 2Go). The deleted region is corresponding to exon 4 of the zebrafish GHRH-PACAP gene (36). The deduced amino acid sequence of zfPACAP38-2 exhibits extremely high homology with that of mammals (92% homology), whereas the sequence of GHRH shows much greater variation from that of other vertebrates. Interestingly, the sequence of zfPACAP38-2 (GenBank accession no. AF329730) is significantly different from zfPACAP38-1 reported previously in the zebrafish in both the nucleotide and amino acid sequences (36); however, the dibasic residues (K129-R130, R159-R160, R170-R171) implicated in posttranslational peptide cleavage to release PACAP are fully conserved. The two forms of zebrafish GHRH-PACAP, GHRH-zfPACAP38-1 and GHRH-zfPACAP38-2, share 69% amino acid homology over the GHRH region and 82% over PACAP (36) (Fig. 2Go); however, the amino acid sequence of zfPACAP38-2 is identical with that of goldfish PACAP38a (32) and shares high homology (92%) with catfish PACAP (33).



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  		FIG. 1. DNA and deduced amino acid sequences of zebrafish ovarian GHRH-PACAP (zfPACAP38-2) (GenBank accession no. AF329730). The potential polyadenylation signal (AATAAA) is underlined in the 3'-untranslational region. The boxed area in the coding region is the sequence deleted in the short spliced form of the transcript. The structure of the full-length cDNA and the cloning strategy are shown in the box at the bottom. Also shown in the box is the PCR product generated and quantitated in the RT-PCR assay.

 


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  		FIG. 2. A, Comparison of the deduced amino acid sequences of the long (L) and short (S) forms of the zfPACAP38-2. The majority of the GHRH molecule is deleted in the short spliced form of the transcript (dashed line). There is an amino acid substitution of serine (S80) for arginine (R80) in the short form because of the differential splicing. B, Amino acid sequence comparison of zfPACAP38-1 (36 ) and zfPACAP38-2.

 
Temporal and spatial patterns of zfPACAP38-2 expression in the zebrafish
To characterize the temporal expression pattern of zfPACAP38-2 in the ovary, we investigated its expression in the developing follicles including previtellogenic (<0.25 mm), midvitellogenic (~0.45 mm), and full-grown immature follicles (~0.65 mm) (Fig. 3Go). Two products of different intensities were amplified from the follicles of all stages as well as cultured follicle cells. The small-product coding for zfPACAP38-2 only was far more abundant than the large one, which codes for both GHRH and zfPACAP38-2; however, whether this difference in amplification reflects the difference in mRNA abundance or is due to different amplification efficiency remains unknown. The expression of zfPACAP38-2 appeared to increase gradually with the development of follicles.



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  		FIG. 3. A, Distribution of zfPACAP38-2 expression in nonovarian tissues. B, Expression of zfPACAP38-2 in the ovary. PV, Previtellogenic follicles; MV, midvitellogenic follicles; IM, full-grown immature follicles; M, full-grown mature follicles; OV, postovulatory ovary; CF, cultured follicle cells; O, whole ovary. +, With RT; -, RNA without RT.

 
We also examined the expression pattern of PACAP in a variety of other tissues including the brain, eyeball, muscle, kidney, gill, liver, and testis. In the brain, eyeball, muscle, and testis, a two-band pattern was also observed despite variation in the relative intensity of the two bands (Fig. 3Go). No product could be detected in the liver, kidney, and gill.

Expression of VPAC2-R in the zebrafish ovary
To provide evidence that PACAP functions as a potential paracrine/autocrine factor in the ovary, we examined whether the zebrafish ovary expresses any PACAP receptor. Using gene-specific primers, a 274-bp cDNA fragment coding for VPAC2-R was cloned from the zebrafish ovary (GenBank accession no. AF329633) and analyzed by sequencing. Similar to zfPACAP38-2, VPAC2-R is also expressed in the follicles of different stages investigated. However, unlike zfPACAP38-2, the expression of VPAC2-R was more or less constant during follicle development. Both RT-PCR and Northern blot analysis revealed a high expression level of VPAC2-R in cultured ovarian follicle cells (Fig. 4Go).



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  		FIG. 4. A, Expression of VPAC2-R in the ovary. PV, Previtellogenic follicles; MV, midvitellogenic follicles; IM, full-grown immature follicles; M, full-grown mature follicles; OV, postovulatory ovary; CF, cultured follicle cells; O, whole ovary. +, With RT; -, RNA without RT. B, Northern blot analysis for VPAC2-R in the cultured follicle cells, whole ovary, and full-grown immature follicles. C, DNA and amino acid sequences of the cloned VPAC2-R fragment.

 
Gonadotropin regulation of zfPACAP38-2 expression in cultured ovarian follicle cells
Gonadotropins are key regulators of the ovary in all vertebrate groups. To examine whether the expression of zfPACAP38-2 and VPAC2-R in the zebrafish ovary is influenced by gonadotropin, we performed the present experiments to investigate their expression in the zebrafish ovary in response to hCG using semiquantitative RT-PCR. The RT-PCR assays were carefully validated by determining the cycle number that generates half-maximal amplification for each gene. The cycle numbers were then further tested by amplifying serially diluted templates to evaluate the correlation between the input of template and the output of PCR amplification (Fig. 5Go). The optimized cycle numbers are 29 for zfPACAP38-2, 27 for VPAC2-R, and 19 for ß-actin (data not shown).



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  		FIG. 5. Validation of the RT-PCR assays for zfPACAP38-2 and VPAC2-R. Upper panel, Kinetics of PCR amplification for zfPACAP38-2 and VPAC2-R. The cycle number that generates half-maximal reaction was used to analyze the expression level of each gene. Lower panel, PCR amplification of cloned zfPACAP38-2 and VPAC2-R cDNA using the cycle number obtained from the upper panel. Each value represents the mean ± SEM of three PCRs, and the electrophoretic image is shown at the bottom of each graph.

 
hCG (15 IU/ml) significantly increased the expression level of zfPACAP38-2 in a clear time-dependent manner. The effect reached maximal level at 2 h of the treatment but gradually diminished with longer treatment (4–8 h). Both the long and short transcripts were increased. When tested at 2 h of the treatment, hCG stimulated zfPACAP38-2 expression in a dose-dependent manner, with the maximal response reached at 15 IU/ml. No desensitization was noticed at a high dose of hCG (50 IU/ml) during the 2 h treatment (Fig. 6Go). In contrast to the response of zfPACAP38-2, the expression of VPAC2-R was constant, with little response to hCG treatment (Fig. 7Go).



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  		FIG. 6. Effects of gonadotropin(s) (hCG and goldfish pituitary extract) on the expression of zfPACAP38-2 in cultured zebrafish ovarian follicle cells. In the time course experiments, hCG (15 IU/ml) (A) or goldfish pituitary extract (30 µg/ml) (C) was applied to the cultured follicle cells for different time periods (0–8 h) before RNA extraction, whereas in the dose-response experiment, the cells were treated for 2 h with different doses of hCG (0–50 IU/ml) (B) or goldfish pituitary extract (0–30 µg/ml) (D). The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCR of three replicates, and the electrophoretic image is shown at the bottom of each graph. **, P < 0.001 vs. control.

 


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  		FIG. 7. Effect of hCG on the expression of VPAC2-R in cultured zebrafish ovarian follicle cells. In the time course experiments, hCG (15 IU/ml) (A) was applied to the cultured follicle cells for different time periods (0–8 h) before RNA extraction, whereas in the dose-response experiment, the cells were treated for 2 h with different doses of hCG (0–50 IU/ml) (B). The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCRs of three replicates.

 
To confirm the regulation of zfPACAP38-2 expression by gonadotropin in the zebrafish ovary, we further examined the effect of pituitary extract prepared from sexually mature goldfish. Similar to hCG, goldfish pituitary extract also strongly up-regulated the expression of zfPACAP38-2 in clear time- and dose-dependent manners. The effect became evident at the concentration of 3 µg/ml and reached the maximal level at 10 µg/ml (Fig. 6Go).

Because cAMP is well known as the major second messenger involved in gonadotropin signaling (41, 42), we also studied the effect of increasing intracellular cAMP level on the expression of zfPACAP38-2. Both cAMP analogs (8-Br-cAMP and db-cAMP) and forskolin (an activator of adenylate cyclase) were used in the experiments. Treatment of cultured zebrafish follicle cells with cAMP analogs or forskolin for 2 h significantly increased the expression of zfPACAP38-2 in a dose-dependent manner (Fig. 8Go).



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  		FIG. 8. Effects of 8-Br-cAMP (A), db-cAMP (B), and forskolin (C) on the expression of zfPACAP38-2 in cultured zebrafish ovarian follicle cells. The cells were treated for 2 h before RNA extraction. D, Effects of hCG (15 IU/ml) and forskolin (FK; 10 µM) on zfPACAP38-2 expression in the absence or presence of H89 (10 µM). The expression levels were normalized by ß-actin and expressed as the percentage of respective control. Each value represents the mean ± SEM of independent RT-PCRs of three replicates. **, P < 0.001 vs. control.

 
To understand the signaling mechanisms downstream of cAMP in the regulation zfPACAP38-2 expression, we assessed the effects of hCG and forskolin in the zebrafish follicle cells in the presence or absence of H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). As shown in Fig. 8Go, H89 (10 µM) completely blocked hCG (15 IU/ml)- and forskolin (10 µM)-stimulated expression of zfPACAP38-2, and it also seemed to decrease the basal level of zfPACAP38-2 mRNA.

Effect of PACAP on the final maturation of zebrafish oocytes
The evidence from the present study and others (32, 33, 36) strongly points to PACAP as an intraovarian regulator in fish; however, no information is available on the roles of this peptide in fish ovarian functions. To provide clues to the functional roles of PACAP in the zebrafish ovary, we first examined its effect on the rate of oocyte maturation, an event that involves multiple endocrine hormones and local paracrine factors. Synthetic zebrafish zfPACAP38-2 at 18 h significantly enhanced the maturation rate (GVBD) of full-grown zebrafish oocytes with a bell-shaped dose-response curve. The maximal effect of zfPACAP38-2 was observed at 1 nM; however, the effect diminished with increasing concentration and completely disappeared at 100 nM or higher. When tested at 1 nM, zfPACAP38-2 had a clear time-dependent effect on zebrafish oocyte maturation, with the effect becoming significant after 8 h incubation (Fig. 9Go). When zfPACAP38-2 (1 nM) was applied together with different concentrations of hCG (0–50 IU/ml) or DHP (0–10 ng/ml), the maturation-inducing hormone in most teleosts, their effects appeared to be additive at the low range of concentration. The disappearance of the additive effects at high concentrations of hCG and DHP is obviously due to the upper response limit of the assay (Fig. 9Go).



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  		FIG. 9. Dose response (A) and time course (B) of zfPACAP38-2 on the final oocyte maturation in the zebrafish, and its interaction with hCG (C) and DHP (D). In all of the dose-response experiments, the follicles were treated for 18 h (A, C, and D). Each value represents the mean ± SEM of three replicates. *, P < 0.05; **, P < 0.001 vs. control of respective time or dose points.

 
PACAP regulation of ovarian follistatin expression in cultured zebrafish follicle cells
Our previous studies have established a critical role for the activin regulatory system in the function and regulation of zebrafish ovary (43, 44, 45, 46, 47). A recent study from our laboratory demonstrated that follistatin, a potent activin-binding protein, was strongly stimulated by gonadotropin via the cAMP-PKA pathway in zebrafish follicle cells (43). Because PACAP signaling also involves cAMP and its VPAC2-R is abundantly expressed by the zebrafish follicle cells, it would be interesting to find out whether PACAP has any effect on the expression of follistatin, which would provide clues to potential links between PACAP and activin system. Thirty cycles of amplification were used in the RT-PCR assay for follistatin, which has been validated in our previous study (43). As shown in Fig. 10Go, zfPACAP38-2 significantly increased follistatin expression in a clear dose-dependent manner. The maximal effect was noticed again at 1 nM, and the response showed a slight trend of decline over the concentration range of 10–1000 nM. We also examined the response of activin ßA and ßB subunits to zfPACAP38-2; however, the results were inconsistent among experiments (data not shown).



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  		FIG. 10. Effect of zfPACAP38-2 on the expression of follistatin in cultured zebrafish ovarian follicle cells. The cells were treated for 2 h with different doses of zfPACAP38-2 (0–1000 nM) before RNA extraction. Each value represents the mean ± SEM of three replicates. **, P < 0.001 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A previous study has cloned and characterized a GHRH-PACAP gene in the zebrafish (36). In the present study, we cloned a cDNA from the zebrafish ovary that codes for a new type of PACAP, zfPACAP38-2. The high level of sequence variation between the two types of PACAP precursors in the zebrafish suggests an early duplication of PACAP gene in the zebrafish lineage, which is likely due to an early genome duplication (48, 49).

Our RT-PCR analysis demonstrated two products of different sizes in both the ovary and several other tissues including the brain, testis, eyeball, and muscle. Sequence analysis of the products revealed that the product of large size encodes both GHRH and zfPACAP38-2, whereas the small one codes only for zfPACAP38-2 and some C-terminal amino acids of GHRH with the majority of the GHRH molecule deleted. Because the deleted region is corresponding to exon 4 of the zebrafish GHRH-PACAP gene (36), the short transcript is likely generated through an alternative mRNA-splicing mechanism. This is different from the previously reported zfPACAP38-1 gene, which generates only one single transcript in the tissues examined (36), but similar to the findings in other nonmammalian species such as Xenopus (37), chicken (38), and several teleost fish (35, 50), suggesting that this expression pattern is well conserved in vertebrates, at least in nonmammalian species. Our results showed that the product amplified from the small transcript was consistently more abundant than the large one in both ovary and nonovarian tissues. Although this may reflect the relative abundance of the two transcripts in the tissues, the possibility of different amplification efficiency for the two products cannot be ruled out.

The expression of zebrafish zfPACAP38-2 in the ovary suggests a local role for this peptide in the regulation of ovarian functions. This is supported by our demonstration that PACAP receptor VPAC2-R is also expressed in the zebrafish ovary. Although the exact location of VPAC2-R expression and therefore the sites of PACAP action in the ovary are not known, the follicle cells are likely the major target for PACAP, because VPAC2-R is most abundantly expressed in the cultured follicle cells. In mammals, RT-PCR analysis and in situ hybridization have demonstrated the expression of all three types of PACAP receptors in the ovary (22, 23, 29, 30, 31), which, together with our evidence, strongly implicates PACAP as a local paracrine/autocrine factor in the regulation of vertebrate ovarian functions.

The development and function of vertebrate ovary are tightly controlled by pituitary gonadotropins. However, increasing evidence shows that the activities of gonadotropins are mediated or modulated by an increasing list of intraovarian paracrine/autocrine growth factors including activin, IGFs, and epidermal growth factor (51, 52). With the evidence increasing for PACAP as a potential local factor, it would be interesting to understand how it is regulated in the ovary. Evidence from the present study showed that zfPACAP38-2 in the zebrafish ovary is strongly up-regulated by hCG and goldfish pituitary extract, consistent with the studies in the rat (19, 20, 21, 24). This suggests that PACAP could serve as a mediator in gonadotropin actions. Consistent with the effect of gonadotropin, cAMP analogs (8-Br-cAMP and db-cAMP) and forskolin, which increase the level of intracellular cAMP, also significantly increased zfPACAP38-2 expression in a dose-dependent manner in cultured follicle cells. The effects of hCG and forskolin could both be completely blocked by H89, suggesting that the up-regulation of zfPACAP38-2 expression by gonadotropin(s) is primarily mediated by activation of cAMP-PKA signaling pathway. These data agree with the presence of two cAMP response elements in the promoter of rat and mouse PACAP gene (53, 54). Because cAMP is also the major second messenger used by PACAP, the strong up-regulation of zfPACAP38-2 by gonadotropin and cAMP suggests that PACAP in the zebrafish ovary either serves as a downstream factor to relay the actions of gonadotropin from one cell type to another in a paracrine manner, or it may act as part of a closed-loop positive feedback mechanism to amplify the actions of gonadotropin in an autocrine manner if it works on the same cells. In contrast to zfPACAP38-2, VPAC2-R in the zebrafish follicle cells did not seem to respond to hCG at the transcriptional level, similar to a recent report in the rat that gonadotropins had no effect on VPAC2-R expression in granulosa cells (22). The powerful stimulatory effect of gonadotropins on PACAP but not VPAC2-R expression in the ovary of both zebrafish and mammals strongly suggests that the ovarian PACAP-mediated paracrine system and its regulation are well conserved, and the system may therefore play fundamental roles in the development and function of the ovary across vertebrates.

PACAP in the zebrafish ovary may participate in the entire process of folliculogenesis, because the transcripts of both zfPACAP38-2 and VPAC2-R were detected in the follicles of all stages examined including previtellogenic, vitellogenic, and full-grown follicles. In mammals, PACAP has been shown to regulate the functions of the large preovulatory follicles, which are probably related to the preovulatory cascade (27, 28). In the present study, we demonstrated that zfPACAP38-2 at low dosage had a stimulatory effect on the final oocyte maturation in the zebrafish, and its effect was additive to those of hCG and DHP over a low concentration range of these hormones; however, the effect of zfPACAP38-2 decreased at high concentrations. The stimulatory effect of zfPACAP38-2 on zebrafish oocyte maturation is consistent with the reports in the rat that PACAP and VIP enhance meiotic maturation of follicle-enclosed oocytes (28, 55). The reason for the loss of zfPACAP38-2 effect at high dosage is unknown. It may be due to receptor desensitization or a direct action of PACAP on the oocytes. It has been shown in the rat that granulosa-luteal cells express PACAP receptors (PAC1-R and VPAC2-R), but the specific PACAP binding sites have also been localized on oocytes (31). The activation of these receptors on oocyte surface by PACAP results in an increase of cAMP level within the oocytes, which delays or prevents oocyte maturation (28, 55, 56).

In addition to oocyte maturation, we also examined the effect of zfPACAP38-2 on the expression of follistatin, which is a potent binding protein of activin, a dimeric protein belonging to the TGF-ß superfamily (57). The reason we were interested in follistatin is that its expression in the zebrafish follicle cells increases dramatically in response to cAMP (43), which is also a major second messenger in PACAP signaling in rat granulosa or granulosa-luteal cells (25, 26, 27, 28, 31, 58, 59). As expected, zfPACAP38-2 significantly increased follistatin expression in the zebrafish ovarian follicle cells in a dose-dependent manner. Although there is no information on PACAP regulation of follistatin in the ovary of vertebrates, PACAP has been shown to stimulate follistatin expression in cultured pituitary cells (18, 60, 61) and enhance follistatin promoter activity in {alpha}T3-1 cells (18). The regulation of follistatin by zfPACAP38-2 in the zebrafish ovary suggests that part of the effects of PACAP in the ovary may be due to its impact on the local activin-follistatin system.

In summary, the present study identified and cloned a new type of PACAP, zfPACAP38-2, in the zebrafish ovary, which is different from zfPACAP38-1 reported before. zfPACAP38-2 is coded by two mRNA transcripts of different sizes generated through differential mRNA splicing. The big transcript encodes both zfPACAP38-2 and GHRH, whereas the small one codes only for zfPACAP38-2. The expression of zfPACAP38-2 in the zebrafish ovary was strongly stimulated by gonadotropin(s) (hCG and goldfish pituitary extract), and the up-regulation could be mimicked by cAMP analogs and forskolin but suppressed by H89, suggesting that the gonadotropin action on zfPACAP38-2 expression is largely dependent on the activation of the cAMP-PKA signaling pathway. The functional roles of PACAP in the zebrafish ovary are substantiated by our evidence that zfPACAP38-2 enhanced final oocyte maturation and stimulated follistatin expression in the follicle cells. These, together with the expression VPAC2-R in the zebrafish ovary, suggest that zfPACAP38-2 is a potential downstream mediator of gonadotropin actions in the ovary, and part of its activities may be due to its influence on the intraovarian activin-follistatin system that is critical in the regulation of vertebrate ovarian functions.


   Footnotes
 
This work was substantially supported by Grants CUHK4176/99M, CUHK4150/01M, and CUHK4258/02M from the Research Grants Council of the Hong Kong Special Administrative Region (to W.G.).

Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; db-cAMP, dibutyryl-cAMP; DHP, 17{alpha},20ß-dihydroxy-4-pregnen-3-one; GVBD, germinal vesicle breakdown; hCG, human chorionic gonadotropin; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; RACE, rapid amplification of cDNA ends; RT, reverse transcription; VIP, vasoactive intestinal peptide; zf, zebrafish.

Received April 21, 2003.

Accepted for publication July 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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